Phosphoinositide 3'-kinases (Pi3Ks) phosphorylate inositol phospholipids normally present in the cell at the 3-OH position of the inositol ring. A growing body of evidence suggests that the resultant 3'-phosphorylated lipids act as second messengers in signalling pathways governing a broad spectrum of cellular processes. A major focus of the research in this field is currently aimed at identifying downstream targets of these inositol phospholipids and elucidating the molecular mechanisms by which they function as second messengers. Pursuant to that goal, the host laboratory has isolated novel proteins from concentrated tissue extracts by virtue of their ability to bind to phosphatidylinositol(3,4,5)-trisphosphate (PtdIns(3,4,5)P3) coupled to agarose beads. Described in this proposal are the approaches that will be undertaken to clone and characterize at least 2 of the novel proteins that have been isolated. The genes encoding the novel proteins will be cloned by screening relevant cDNA libraries with oligonucleotide probes based on tryptic peptide sequence that has already been obtained. The affinity and specificity of purified recombinant proteins to a panel of phospholipids will be examined. The ability of the proteins to translocate to the membrane in vivo in a PI3K-dependent fashion will also be investigated. Finally, proteins with which the novel proteins interact will be identified, in an endeavor to define the signalling pathways in which they participate. The information obtained from these studies will further our understanding of P13K signal transduction pathways, and may offer promising targets for therapeutic intervention in the treatment of human disease.